The 28-year-old, who left the Saints at the end of the 2012-13 campaign, spent the second part of last season with the Lions.
Injury forced the midfielder to return to his parent club, but Chaplow has now agreed a permanent deal with the Championship outfit.
"I enjoyed my time here and when the option of joining the club permanently came up, it was one I was keen to accept," he told the club's official website.
"I'm looking forward to getting back out on the training ground with the boys and picking up where I left off."
Chaplow has become Millwall's fifth signing of the summer.